Glucose-6-phosphate dehydrogenase activity in monolayer cultures of thyroid epithelial cells: TSH and inhibition of nitrogen oxide synthase affect the enzyme activity and the oxygen sensitivity of the histochemical assay

The objective was to investigate glucose-6-phosphate dehydrogenase (G6PD) activity in monolayer cultures of thyroid epithelial cells and to examine whether inhibition of nitric oxide synthase affects activity of G6PD or oxygen sensitivity of the assay. Primary cultures without TSH addition prior to experiments demonstrated a TSH-dependent increase in G6PD activity. G6PD activity was higher in F12 medium than in a serum-free physiological medium. Secondary cultures grown in F12 medium demonstrated a diminished activity of G6PD and a lack of response to TSH. In the serum-free physiological medium, G6PD activity was comparable to that found in primary cultures and a response to high concentrations of TSH was maintained. In primary cultures grown in F12 medium devoid of TSH, G6PD activity decreased dose-dependently when nitric oxide synthase activity was inhibited. The oxygen sensitivity of the assay was comparable to that reported previously in malignant cells and correlated with the activity of G6PD in primary cultures. We suggest that thyroid epithelial cells may be an appropriate system to investigate oxygen sensitivity of the G6PD assay as the cells demonstrate a reduced oxygen sensitivity which can be influenced by culture conditions.     

Characterization of the osmotic-stress response inSaccharomyces cerevisiae: osmotic stress and glucose repression regulate glycerol-3-phosphate dehydrogenase independently

Micro-organisms have developed systems to adapt to sudden changes in the environment. Here we describe the response of the yeastSaccharomyces cerevisiae to osmotic stress. A drop in the water activity (a_w) of the medium following the addition of NaCl led to an immediate shrinkage of the cells. During the 2 h following the osmotic shock the cells partially restored their cell volume. This process depended on active protein synthesis. During the recovery period the cells accumulated glycerol intracellularly as a compatible solute and very little glycerol was leaking out of the cell. We have investigated in more detail the enzymes of glycerol metabolism and found that only the cytoplasmic glycerol-3-phosphate dehydrogenase was strongly induced. The level of induction was dependent on the yeast strain used and the degree of osmotic stress. The synthesis of cytoplasmic glycerol-3-phosphate dehydrogenase is also regulated by glucose repression. Using mutants defective in glucose repression (hxk2), or derepression (snf1), and with invertase as a marker enzyme, we show that glucose repression and the osmotic-stress response system regulate glycerol-3-phosphate dehydrogenase synthesis independently. We infer that specific control mechanisms sense the osmotic situation of the cell and induce responses such as the production and retention of glycerol.     

Post-exercise ketosis and the glycogen content of liver and muscle in rats on a high carbohydrate diet

Summary Post-exercise ketosis is known to be suppressed by physical training and by a high carbohydrate diet. As a result it has often been presumed, but not proven, that the development of post-exercise ketosis is closely related to the glycogen content of the liver. We therefore studied the effect of 1 h of treadmill running on the blood 3-hydroxybutyrate and liver and muscle glycogen concentrations of carbohydrate-loaded trained (n=72) and untrained rats (n=72). Resting liver and muscle glycogen levels were 25%–30% higher in the trained than in the untrained animals. The resting 3-hydroxybutyrate concentrations of both groups of rats were very low: <0.08 mmol·1⁻¹. Exercise did not significantly influence the blood 3-hydroxybutyrate concentrations of trained rats, but caused a marked post-exercise ketosis (1.40±0.40 mmol·1⁻¹ 1 h after exercise) in the untrained animals, the time-course of which was the approximate inverse of the changes in liver glycogen concentration. Interpreting the results in the light of similar data obtained after a normal and low carbohydrate diet it has been concluded that trained animals probably owe their relative resistance to post-exercise ketosis to their higher liver glycogen concentrations as well as to greater peripheral stores of mobilizable carbohydrate.     

Growth resistance-sized arteries in response to bladder hypertrophy in the rat: time-course,DNA-synthesis and LDH-isoform pattern

Bladder growth was induced by partial urethral obstruction. Bladder hypertrophy was evident at 53 h after obstruction and continued over a 6 weeks period. Small bladder arteries were taken from fixed anatomical locations of the bladder circulation, mounted in a small vessel myograph and the optimal diameter for maximal isometric force development was determined (L_max, K⁺=125 mm stimulation). Bladder hypertrophy was associated with an enlarged L_max from 53 h onward (compared with sham-operated controls) and L_max continued to increase until 10 days after urethral obstruction. Between 10 days and 6 weeks no further increase of the diameter was observed. Increased diameters in vitro were accompanied by a transiently increased [³H]Thymidine uptake in the small arteries which peaked at 53 h after obstruction but was still above background at 10 days. At this time point, small arterial growth was associated with a significant relative increase in the M isoform of LDH as determined with agarose electrophoresis on tissue homogenates. Thus organ growth induced small vessel growth in the rat is characterized by a rapid onset, increased but transient DNA-turnover and LDH-isoform changes. The latter mimic changes seen in other types of smooth muscle growth.     

Changes in glutamate-related enzyme activities in the striatum of the rat following lesion of corticostriatal fibres

Summary The behaviour of enzymes putatively involved in glutamate/aspartate transmitter metabolism (glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase,-glutamyltranspeptidase) was studied in the striatum 3, 7, 14 days and 7 weeks after mechanical destruction of corticostriatal fibres. For a period of up to seven days after unilateral lesion, enzyme activities were significantly diminished (by up to 13% based on protein) in the ipsilateral striatum as compared to the striatum of the intact side. Later, the enzyme activities in the ipsilateral striatum recovered. After seven weeks, an increase was observed for glutamate dehydrogenase activity, whereas the activity of alanine aminotransferase showed a transient rise at the end of the second week. The decrease in enzyme levels is interpreted as being attributable to the destruction of nerve endings which are considered to be glutamatergic, interfering with various compensating processes (e.g. glial cell proliferation) which occur with advancing times after lesion.     

Calretinin- and parvalbumin-immunoreactive neurons in the rat main olfactory bulb do not express NADPH-diaphorase activity

The presence of nitric oxide synthase (NOS) in neuronal elements expressing the calcium-binding proteins calretinin (CR) and parvalbumin (PV) was studied in the rat main olfactory bulb. CR and PV were detected by using immunocytochemistry and the nitric oxide (NO) -synthesizing cells were identified by means of the reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) direct histochemical method. The possible coexistence of NADPH-diaphorase and each calcium-binding protein marker was determined by sequential histochemical-immunohistochemical double-labeling of the same sections. Specific neuronal populations were positive for these three markers. A subpopulation of olfactory fibers and olfactory glomeruli were positive for either NADPH-diaphorase or CR. In the most superficial layers, groups of juxtaglomerular cells, superficial short-axon cells and Van Gehuchten cells demonstrated staining for all three markers. In the deep regions, abundant granule cells were NADPH-diaphorase- and CR-positive and a few were PV-immunoreactive. Scarce deep short-axon cells demonstrated either CR-, PV-, or NADPH-diaphorase staining. Among all these labeled elements, no neuron expressing CR or PV colocalized NADPH-diaphorase staining. The present data contribute to a more detailed classification of the chemically- and morphologically-defined neuronal types in the rodent olfactory bulb. The neurochemical differences support the existence of physiologically distinct groups within morphologically homogeneous populations. Each of these groups would be involved in different modulatory mechanisms of the olfactory information. In addition, the absence of CR and PV in neuronal groups displaying NADPH-diaphorase, which moreover are calmodulin-negative, indicate that the regulation of NOS activity in calmodulin-negative neurons of the rat olfactory bulb is not mediated by CR or PV.     

Alteration of regulatory enzyme activities in fast-twitch and slow-twitch muscles and muscle fibres in low-intensity endurance-trained rats

The effect of progressive, low-intensity endurance training on regulatory enzyme activities in slow-twitch (ST) and fast-twitch (FT) muscle fibres was studied in 32 rats. Of those rats 16 were trained on a treadmill at a running speed of 10m · min^–1 5 days a week over an 8-week period. Running time was progressively increased from 15 min to 2 h · day^–1. Of the rats 4 trained and 4 sedentary rats were also subjected to acute exhausting exercise. Enzyme activities of phosphofructokinase 1 (PFKI) from glycolysis, -ketoglutarate dehydrogenase (-KGDH) from the Krebs cycle and carnitine palmitoyltransferase (CPT I and II) from fatty acid metabolism in soleus, tibialis anterior and gastrocnemius muscles were measured in trained and sedentary rats. Enzyme activities of individual ST and FT fibres were measured from the freeze-dried gastrocnemius muscle of 8 trained and 8 sedentary rats. In the sedentary rats the activity of PFK1 in tibialis anterior and soleus muscles was 141% and 41% of the activity in gastrocnemius muscle, respectively. The activity of -KGDH in tibialis anterior and soleus muscles was 164% and 278% of the activity in gastrocnemius muscle, respectively. The activity of CPT I in tibialis anterior and gastrocnemius muscles were at the same level, but in soleus muscle the activity was 127% of that in mixed muscle. Endurance training increased enzyme activities of -KGDH and CPT I significantly (P < 0.05) in gastrocnemius muscle but not in soleus or tibialis anterior muscle. After training both -KGDH and CPT II activities were elevated significantly (P < 0.05) in the ST fibres of gastrocnemius muscle, whereas in FT fibres only -KGDH was increased. For PFK1 activity no significant change was observed in ST or FT fibres. After acute exercise, activities of mitochondrial enzymes -KGDH and CPT I tended to be elevated in all muscles. Thus, low-intensity endurance training induced significant peripheral changes in regulatory enzyme activities in oxidative and fatty acid metabolism in individual ST or FT muscle fibres.     

NMDA receptor mediated dendritic plasticity in cortical cultures after oxygen-glucose deprivation

Dendrites and spines undergo dynamic changes in physiological and pathological conditions. Dendritic outgrowth has been observed in surviving neurons months after ischemia, which is associated with the functional compensation. It remains unclear how dendrites in surviving neurons are altered shortly after ischemia, which might reveal the mechanisms underlying neuronal survival. Using primary cortical cultures, we monitored the dendritic changes in individual neurons after oxygen-glucose deprivation (OGD). Two to four hours of OGD induced approximately 30–50% cell death in 24 h. However, the total dendritic length in surviving neurons was significantly increased after OGD with a peak at 6 h after re-oxygenation. The increase of dendritic length after OGD was mainly due to the sprouting rather than the extension of the dendrites. The dendritic outgrowth after 2 h of OGD was greater than that after 4 h of OGD. Application of NMDA receptor blocker MK-801 abolished OGD-induced dendritic outgrowth, whereas application of AMPA receptor antagonist CNQX had no significant effects. These results demonstrate a NMDA receptor-dependent dendritic plasticity shortly after OGD, which provides insights into the early response of surviving neurons after ischemia.